Briefly, fresh bacterial suspensions were prepared in
tryptone soya broth from overnight cultures and adjusted
to OD600. Individual wells were filled with 0.1 mL aliquots
of the diluted culture. Following overnight incubation at
37 °C without agitation, plates were gently washed with
phosphate buffered saline (pH 7.4) and stained with 100 μL
of 0.1% crystal violet for 30 min at room temperature. Excess
crystal violet was removed by washing, and biofilm was
then quantified by measuring the corresponding OD570 nm of
the supernatant following the solubilisation of crystal violet
in 0.15 mL of 95% ethanol. For each clinical strain tested,
biofilm assays were performed in triplicate and the mean
biofilm absorbance value was determined.